Faithful duplication of the genome requires regulation of replication forks that stall at numerous template blockages. Failure to assist stalled replication forks can lead to incomplete replication and many types of genetic alterations underlying DNA fragility syndromes and tumorigensis. Non-histone proteins tightly bound to DNA (protein barriers) are a major cause of fork blockade, and a large portion of these are located inside the repetitive ribosomal DNA (rDNA). rDNA organizes nucleoli and constitutes 10-30% of the genome across species. As such, rDNA replication influences overall genomic stability as well as RNA and protein synthesis. rDNA protein barriers have unique features such as greater topological stress due to high levels of rRNA transcription and requirement of extended maintenance of the replisome. Mechanisms that can ensure rDNA replication completion given these challenges are unclear. Excitingly, our recent data in yeast suggest that the conserved eight-subunit Smc5/6 complex provides an integrated solution for coping with unique challenges at rDNA. We found that Smc5/6 is essential for completing replication at rDNA but not at non-rDNA regions. We further determined that Smc5/6 limits replication fork reversal at rDNA protein barriers. Our new data let us propose that Smc5/6 uses the combined activities of its subunits to regulate stalled forks at rDNA protein barriers and ensure proper rDNA replication termination. We plan to test this central hypothesis using a combination of molecular, genetic, and biochemical approaches in Aim 1. When stalled replication forks fail to recover, collapsed forks and unreplicated DNA gaps can be repaired by homologous recombination, generating recombination intermediates such as Holliday junctions. Promptly resolving these structures is critical for preventing DNA entanglement during mitosis, which can lead to anaphase bridges, micronuclei formation, and genomic instability. Studies from us and others have uncovered multiple regulatory factors that are critical for Holliday junction removal. However, their functional mechanisms remain to be elucidated. Our current research on one of the conserved regulatory factors, the Esc2 protein, which is critical for genomic stability, leads to new models for its functional mechanisms. In particular, we suggest that Esc2 uses a bimodal strategy for enhancing HJ dissolution, including both a structural contribution and a SUMO-mediated mechanism. In Aim 2, we plan to test this model and define how HJ clearance is enabled by Esc2. To accomplish the goals in this proposal, we will use high-resolution assays in the highly effective yeast system. Outcomes of this proposed work will expand our view of several processes, including how rDNA replication completion is achieved, how replication fork is regulated in a context-specific manner, and how recombination intermediate removal can be assisted by regulatory proteins. As these processes are intimately linked to DNA damage syndromes and cancers, our studies will inform mechanisms underlying these diseases, and help to develop new diagnostic and treatment strategies.